Interferon is a type of important familial cytokine with broad-spectrum anti-virus, anti-cell proliferation and immune regulation. Interferon of mammals can be divided into interferon α, β, γ, ω, etc, wherein IFN-α can be further divided into more than ten kinds of subtypes. According to substantial clinical researches, α type interferon is an important anti-virus and anti-tumor drug. At present, there are mainly rhIFN α1b, α2a and α2b that are most widely used in clinic in China.
In addition, studies have shown that type I family of human IFN-α have more than 20 genes as its members, most of which encode functional proteins and have about 90% homology with each other at the nucleotide level. A considerable number of interferon derivatives or analogues can be produced by means of genetic engineering. At present, the most notable one is Infergen (INFERGEN®, IFN-Con 1), a wholly novel protein engineering drug, which was designed by Amgen, a United States company, based on gene sequence homology of 13 kinds of α type interferons, and approved to be listed by U.S. FDA in 1997 for the treatment of hepatitis C with anti-viral activity 5-10 times as high as interferon α2b. CN 1511849A (applicant: BEIJING TRI-PRIME GENETIC ENGINEERING CO., LTD) disclosed a variety of interferon α family molecules which were obtained by in vitro homologous recombination method and had greater advantages in activity and stability than Infergen.
However, no matter interferon α1b, α2a, α2b, or Infergen, as a protein drug, are all restricted in the clinical treatment because of poor stability, high plasma clearance, short in vivo half-life, easy to produce antigen-antibody reactions, etc. Genetic engineering techniques make large-scale synthesis of recombinant proteins become possible and greatly solve the immunogenic problems induced by heterologous proteins, but still can not overcome such disadvantages as quick plasma clearance and low bioavailability. The results of such disadvantages are as follows: frequent injections of interferon are required to achieve the effective therapeutic concentration in plasma. Moreover, after each injection greater volatility of blood concentration is caused along with formation of crest and trough value of drug concentration, thus possibly increasing the cost of treatment and the risk of drug administration inconvenience and adverse reactions. Therefore, attempts have been made to adopt a variety of drug delivery technology (Drug Delivery Technology) to enhance the efficacy of protein drugs. At present, among drug delivery technologies the most widely studied is pegylation technology (PEGNOLOGY).
Pegylation technology of protein is newly developed in the last decade for improving in vivo pharmacokinetic properties of protein-type drugs. It makes activated polyethylene glycol molecule [Poly(ethylene glycol), PEG] bonded to the surface of the protein molecules, thus affecting the spatial structure of proteins, eventually leading to changes in a variety of biochemical properties of the proteins, such as increased chemical stability, improved capacity of resistance to protease hydrolysis, reduction or disappearance of immunogenicity and toxicity, prolonged in vivo half-life, decreased plasma clearance and so on.
PEG component is an inert long-chain amphiphatic molecule generated by polymerization of ethylene monomers. Now a wide variety of PEG molecules are available. Active functional group of activated PEG can be linked to the special position of treatment molecules (such as amine, thiol or other nucleophilic substances). In most cases, covalent bonding of PEG derivatives can be achieved by using the amino group of lysine and N-terminal of peptide molecules as modification sites, each linking part determines a different isotype. During drug research and development process, PEG isotype distribution is of great significance. Because the biological activity of products has a close relation with specific isotype distribution mixtures, the products must be defined in accordance with distribution requirements. It must be proved that PEG isotype distribution is consistent during the entire drug development process including changing production process and proportional lofting. In the actual production, it brings great difficulties to process control and quality evaluation of the products.
The above trouble can be avoided by site-directed pegylation. More and more attention has been paid to pegylating specific protein sites because it can yield highly specific pegylated products and can effectively control the purity of the modified products, making the process more simple and the product quality much easier to evaluate. Highly selective pegylation of proteins can be performed by use of intramolecular cysteine (Cys) sites. There are few proteins with free sulfhydryl, but the sulfhydryl is an important covalent bond to maintain the spatial structure of proteins. Chemical modification at this site often leads to greater damage to molecular structure, thus losing protein activity. Means using genetic engineering can achieve this purpose. It is worth noting that different proteins or peptides differ in structure and property, as well as what can be introduced and where PEG modifier can be introduced. Cys artificially increased through genetic engineering means will lead to intramolecular mismatch or intermolecular combination, causing molecular instability or the formation of irregular polymer. In this regard, a comprehensive sequence analysis and accurate simulation of the molecular structure will provide a line of thought.
Sequence analysis found that the vast majority of IFN-α molecules including interferon α2a, α2b and IFN-con 1 have four cysteines, wherein there are disulfide bond formed between Cys at position 1 and 99, 29 and 139. The present applicant obtains a new type of interferon (MIFN) through in vitro homologous recombination which also maintained this characteristic. IFN-α1b and the other a family interferon obviously differ in the structure, the former has the fifth Cys at position 86 apart from the above four Cys that form normal disulfide bond. Small trial found that site-directed pegylation of interferon can be carried out by use of the site. Thus, in combination with the characteristics, pegylation of other IFN-α is expected to produce the same result.